CD4saurus Rex & HIVelociraptor vs. development of clinically useful immunological markers: a Jurassic tale of frozen evolution.

C O M M E N T A R Y

Open Access

CD4saurus Rex &HIVelociraptor vs. development of

clinically useful immunological markers: a Jurassic

tale of frozen evolution

Andrea De Maria

and Andrea Cossarizza

Abstract

One of the most neglected areas of everyday clinical practice for HIV physicians is unexpectedly represented by

CD4 T cell counts when used as an aid to clinical decisions. All who care for HIV patients believe that CD4+ T cell

counts are a reliable method to evaluate a patient immune status. There is however a fatalistic acceptance that

besides its general usefulness, CD4+ T cell counts have relevant clincal and immunological limits. Shortcomings of

CD4 counts appear in certain clinical scenarios including identification of immunological nonresponders,

subsequent development of cancer on antiretroviral teatment, failure on tretment simplification. Historical and

recently described parameters might be better suited to advise management of patients at certain times during

their disease history. Immunogenotypic parameters and innate immune parameters that define progression as well

as immune parameters associated with immune recovery are available and have not been introduced into

validation processes in larger trials. The scientific and clinical community needs an effort in stimulating clinical

evolution of immunological tests beyond

“CD4saurus Rex” introducing new parameters in the clinical arena after

appropriate validation

Keywords: CD4+T cells, immune reconstitution, antiviral treatment, clinical trials

Introduction

Basic biomedical research is crucial for understanding

pathogenetic mechanisms of diseases, as well as to

develop new techniques drugs or concepts aimed at

improving patient clinical care. Clinical implications of

basic research are regularly reported in major journals

in an effort to improve the transfer of benchwork into

bedside clinical practices[1], and whenever promising

steps in basic research fail to be introduced, this is

underscored [2].

After the identification of CD4+ T cells as the main

target HIV replication, scientists focused on several

important issues, including pathogenesis of mucosal

infection[3-8], clearance of residual replication[9-13],

evaluation of the involvement of innate immunity in

dis-ease progression[14-16], and identification of

immunolo-gical correlates of protection for vaccine studies[17-20].

New cell subsets, receptors, cytokines, and signaling

pathways were described [16,21-25], and widely available

techniques for the

ex vivo study of cells of the innate

immune system (e.g. pDC, mDC, NK cells, NKTcells) or

of Toll-like receptors were introduced. Models of HIV

pathogenesis have been upgraded to account for and

adjust to the new players and their functional

character-istics. By

“CD4” we now define at least 5 different CD4+

T-cell lineages, central and peripheral memory cells

with variable effector functions (Figure 1). Evolution of

our understanding of CD4+ T-cell type and function

had however little impact on the Jurassik Park of clinical

HIV care and antiretroviral trials. In the majority of

cases, indeed,

“immunology” is still represented by

“quantitative determination of CD4+ T-cell numbers”

alone to assess the immune status of routine patients

attending HIV clinics.

CD4saurus Rex: a relic from old times

For some reason, apparently unexplained factors

prevented our clinical practice from evolving moving

* Correspondence: de-maria@unige.it

1

Centro di Eccellenza per la Ricerca Biomedica, Università di Genova, Genova, Italy

Full list of author information is available at the end of the article

© 2011 De Maria and Cossarizza; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

from discussing patients in terms of CD4+ T-cell counts

alone to including additional specific tests. Clinical

activity and knowledge has evolved rapidly in the same

field leading to inclusion in everyday clinical life of

DExa, PK/PD, ultrasensitive viral load, phenotypic and

genotypic resistance evaluation. When it however comes

to immunology, clinicians stick to a very successful but

Jurassic test like CD4+ T-cell counts, which for the

moment we could nickname

“CD4saurus Rex“. Unlike

real Dinosaurs, CD4+ cell count and its clinical use has

neither evolved/upgraded nor vanished each time a

clin-ician is assessing a patient immune status before taking

critical decisions with HIV patients.

There are few doubts that absolute CD4+ T cell

counts have served, and still serve, as a very robust

sur-rogate for progression to AIDS or death. They correlate

generally well to the level of immune competence of

patients with[26,27] or without[28] HIV infection, and

also in children [29].

Proof of the robustness of this relic of

immunologi-cal evolution is represented by its extensive use as a

surrogate marker of immune competence to stratify or

select patients in all recent trials evaluating or

licen-sing the use of newer drug classes or ART regimens

and in studies assessing the optimal time for ART

initiation.

Even in the quest to identify additional risk factors for

HIV-associated non-AIDS defining diseases, including

nephropathy of cardiovascular disease, CD4+ T cell

counts are the only immune parameter used to stratify

patient cohorts[30,31].

Shortcomings of CD4saurus Rex

The concept of CD4+ counts as clinical surrogate

mar-kers is still valid today and should continue to be used

in routine patient follow-up. However, there are areas of

clinical experience where it falls short of our needs.

Opportunistic infections may be sometimes observed

in patients presenting with CD4+ T cell counts well

above the critical range usually referred to for a given

infection, as is the case for PML, TB[32-34], lymphoma

[35] or Kaposi sarcoma patients who may unexpectedly

present with relatively high CD4+ T cell numbers in the

300-400/μl range[36]. Conversely, at any given CD4+

cell count stratum, a fraction of patients have chances

to develop PML, TB, lymphoma or KS, but CD4+ cell

counts alone do not help us to further narrow down our

attention on those who will actually develop disease. A

surprisingly sustained incidence of HPV-associated

pathology (e.g., cervical carcinoma, anal carcinoma) is

observed even after successful ART with rising CD4+

cell counts[37,38]. Drug simplification to

lopinavir/rito-navir monotherapy is effective at 96 weeks, but only in a

subset of patients (47%) not identified by CD4

+standards at baseline.[39].

These events are apparently unexplained to the

clini-cian each time they are observed and are usually

accepted as one would accept a rainy day. Other factors

in the immune playground are likely to be involved, but

“CD4saururs Rex“ still grabs center stage, and we are

missing part of the picture.

Treatment interruptions guided by the number of

CD4+ T cells (CD4+GTI) have been lately discouraged

by studies that showed significant risk of disease

pro-gression to AIDS or death or of cardiovascular events in

patients selected on a narrow and low range of CD4+ T

cells (i.e.350-200 CD4+/

μl) [40]. Other studies however

show that CD4+GTI is feasible and bears negligible/no

risks for patients with different characteristics and

con-siderably higher CD4+ cell counts (500-750/μl) [41,42].

Analysis of progressing patients on CD4+GTI shows

that other phenotypic or functional immune variables

beyond CD4+ cell counts (e.g.CD4+ nadir, NK cell

phe-notype[43]) may define the subset of patients for whom

STI of CD4+GTI could be an option.

A similar issue is represented by immunological

discordance on ART, which is observed in about 15-20%

of drug-naïve patients starting ART[44]. Patients and

physicians may develop anxiety over failure to recover

CD4+ cells, over prophylaxis and over the risk of

devel-oping AIDS.

Thus, at the single patient level, which means our

everyday life, we have to cope with an absolute number,

without any other parameter that could help explain

outliers, aid individualized management optimization or

help to predict - or at least express a likelihood

Figure 1 Possible definitions of CD4+ T-cells based on current knowledge. CD4+ cell counts only represent sums of individual subsets and do not reflect actual composition. Patients with equal CD4+ cell counts may reflect different proportional composition with possibly widely diverging functional immune characteristics.

-whether a given patient will or will not develop an

unwanted condition/disease course.

What failed on the path of CD4+saurus Rex evolution,

and which options are available

The reasons underlying this

“frozen evolution” of

immu-nological tests applied to HIV patients are multifaceted.

Besides the outstanding robustness in terms of wide

availability, standardization, and quality control of

“CD4saurus Rex“, there are few additional assays that

raise some interest and that have been proposed as an

additional qualitative or quantitative measure of the

like-lihood for a patient to develop a specific pattern of

pro-gression or of response to a given treatment.

The drive to improve basic understanding of HIV

associated immune derangements could play a role in

leaving little time and money to refine clinical use of

acquired experience. Competition and a limited

propen-sity of different researchers to integrate techniques

developed elsewhere into clinical practice, also may play

a role in the lack of translation of benchwork into

bed-side assets for patients. The misleading perception that

budget restraints in immune evaluations are justified,

particularly in the absence of wide integration of

immu-nology centers, leaves us with large studies where CD4+

T-cells are the only immune measure, preventing

inclu-sion of additional tests (Figure 2).

Alternative use of qualitative CD4+ T cell analysis,

instead of CD4+ T cell count alone, has been proposed

for other infectious diseases. It is well known that

changes in the phenotype of CD4+ T cells occur in a

large number of viral infections, and can be easily

moni-tored. For example, atypical lymphocytes expressing

CD4+/CD45RO+ may play the role of helper T cells in

the development of the mononucleosis-like syndrome

which is associated with Hepatitis-A infection[45].

During Epstein-Barr infection, a high number of CD4

+Foxp3+ Treg cells can be localized in tonsils, which

are the port of entry of the virus [46], and their role in

inhibiting CD8+ T cell activity is under investigation.

Recently, flow cytometry has also revealed its utility in

providing informative patterns that can differentiate

between infections of bacterial and viral origin. Indeed,

these patterns have been obtained by combining the

fractions of HLA-DR expressing T cell subpopulations

with the level of CD40 on monocytes [47].

Discussion

An improved approach to clinical development of

immune measures should be designed and validated in

the HIV arena. Embedding promising - or rather

con-firmed -immune parameters in new phase III/IV trials

for ART or management optimization would provide a

stimulus for the validation of additional clinical tools

(Table 1). So far, no large study has tried to validate any

of known potential markers which would add clinical

information to CD4+ T-cell counts. These tests,

includ-ing expression of CD38 or CD127, degree of T or NK

cell activation (e.g.:HLA-DR, CD69 or Ki67), NCR

expression by NK cells, or KIR:HLAtyping, could be

used to flag different clinical options or outcomes at

dif-ferent times of the disease/treatment course (e.g.:

immune-reconstitution, ART switch to monotherapy in

selected patients or CD4+GTI, surveillance for

unex-pected opportunistic events including AIDS defining

and non-AIDS defining neoplasms).

In industrialized countries, most laboratories are

equipped with flow cytometers that are able to analyze

routinely multiple (at least 4, up to 8) fluorescent

cellular markers. More sophisticated approaches based

on polychromatic flow cytometry have allowed to

iden-tify a relevant heterogeneity within the CD4+ T cell

compartment [48]. Additional information on the

immunological status of a given patient can be easily

obtained, and should be customized on patient needs.

Testing would not be required on a regular

“routine”

basis, but rather could be applied just before a

“strate-gic management decision

”, to estimate the likelihood

of a given patient or patient group to have different

clinical outcomes. This would contain costs and

pro-vide optimal use of a dedicated test. For example,

con-cerning management of a drug naïve patient, one of

the main questions would be whether the patient will

become an immunological non-responder. Thus, tests

for assessing the capacity of producing new T-cells, in

terms of thymic functionality (such as the amount of

TREC+cells, IL-7 plasma levels, expression of CD127)

[49-51] could be introduced. Similarly, KIR:HLA

car-riage and IL-28 Bpolymorphisms condition significantly

treatment response during HCV infection[52-54] and

has implications also on disease course in coinfected

patients and possibly bear on response to ART[55-58].

Concerning an advanced patient failing a drug

regi-men, markers of CD8+ T cell activation (such as

CD38, CD95 or MHC class II), differentiation

(CD45RA, CCR7 or CD62L), survival (CD127) and of

CD4 activation and differentiation could be crucial

(Table 1). Similar considerations could be applied to

successfully treated patients with suppressed VL and

recovered CD4+ T cell count, who would candidate for

simplification regimens (or for possible drug vacation

on the basis of NK activating/inhibitory receptor

phe-notype[43] (Table 1). Last but not least, age-related

immunological changes in a huge number of

para-meters, including the subpopulations of CD4+ T cells,

have to be considered when

“normal” levels of a

bio-marker are studied, considering that the aging of HIV+

patients is an emerging problem of relevant

impor-tance (50).

Conclusion

In conclusion, time has come to introduce

complemen-tary customized parameters, in addition to CD4+ cell

counts, in the clinical care of HIV-infected patients in

order to provide additional immuno-virological

stratifi-cation criteria. This may be achieved by specifically

investing on appropriate validation/standardization

strategies in clinical trials (extending in range from ART

initiation to ART optimization) using available

parameters

Table 1 List of useful or promising analyses, in addition to

CD4+saurus Rex testing, so far unaccounted for in clinical

trial validation but potentially relevant in every-day patient management and clinical decisions

What to test Who and When Possible use/interpretation Ref.

PBMC

PD1+_DR+_Ki67+_CD4+

Ki67+PD-1+CD4+Treg Th17 cytokine profile

Before cART in adv.naive and AIDS-presenter pts

Increase. Predict likelihood of IRIS. Diagnosis of IRIS (uppon symptoms

[59]

Plasma TNF-a, 4, IL-17, VEGF,

G-CSF, GM-CSF, CCL2 (MCP-1)

pt.with cryptococcal meningitis Increase. Predicts high risk of IRIS [60]

PBMC

CD56bright NK cells NKp30+CD56+, NKp46 +CD56+

NK cells

Before Voluntary or CD4+guided Treatment interruption

Increase. Advise against interruption for risk of rapid CD4 decrease when markers are increased

[43]

PBMC

CD4+/62L+/RA+ CD8+/CD38+/DR+ CD8+/62L+/RA+

Wk16-24 of cART - Adolescents Increase. Risk of Virological Failure after initial response [61]

PBMC

HLA-Bw4 (incl.HLA-B*57, HLA-B*27)

HIV infection, At first diagnosis Presence. Defines lower risk of progression, chances of Elite Controlling, slow progression, lower VL

[62-65]

PBMC

HLA-B*57 + KIR3DS1

Exposed uninfected partners, Any time Presence. Decreased risk of infection upon HIV exposure [66-68] PBMC

HLA-B*57 + KIR3DL1high

Exposed uninfected partners, Any time Presence. Decreased risk of infection upon HIV exposure [69]

HLA-B*57 HIV-Infected, Before cART start Presence. Defines adverse reaction to Abacavir [70]

CCR5-∂32, CCR2-64I At diagnosis. Presence. Slower disease progression, lower VL [71]

Acknowledgements

This work has been supported in part by grants awarded by Istituto Superiore di Sanita (I.S.S., Programma Nazionale AIDS n. 40G.41/40F.55 and 45G.11), Accordi di Collaborazione Scientifica n. 40D61, 40H69 and 45D/1.13 Author details

1_{Centro di Eccellenza per la Ricerca Biomedica, Università di Genova,}

Genova, Italy.2Dipartimento Scienze della Salute (DISSAL), Università di Genova, Italy.3_{S.S. Infettivologia, Istituto Nazionale per la ricerca sul Cancro,}

Genova, Italy.4Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Modena, Italy.5_{Departamento de Bioquímica y Biología}

Molecular, Universidad de Valencia, Valencia, Spain. Authors_{’ contributions}

ADM conceived the design of the commentary, discussed and wrote the manuscript. AC participated in the design of the commentary, discussed and wrote the manuscript. All authors read and approved the final manuscript Competing interests

The authors declare that they have no competing interests. Received: 19 April 2011 Accepted: 16 June 2011 Published: 16 June 2011

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doi:10.1186/1479-5876-9-93

Cite this article as: De Maria and Cossarizza: CD4saurus Rex &HIVelociraptor vs. development of clinically useful immunological markers: a Jurassic tale of frozen evolution. Journal of Translational Medicine 2011 9:93.

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